青钱柳愈伤组织的离体保存

In vitro Conservation of Callus of Cyclocarya paliurus

【目的】在优化植物激素的基础上,通过添加渗透调节剂和生长抑制剂,探索青钱柳愈伤组织离体保存的方法,为青钱柳种质资源的长期离体保存提供理论依据和技术支撑。【方法】以青钱柳愈伤组织为试验材料,采用二因素完全随机法优化培养基内6-BA(0.4、0.6、0.8、1.0 mg·L-1)+IBA(0.2、0.4 mg·L-1)浓度组合;在此基础上,采用单因素完全随机法,针对不同蔗糖浓度(30、45、50、55、60 g·L-1)及其保存时间(60、90、120、150、180天)、不同渗透调节剂浓度(甘露醇:0、10、20、30、40、50 g·L-1;肌醇:0、10、20、30、40、50 g·L-1)及不同生长抑制剂浓度(多效唑(PP 333):0、2、4、6、8、10 mg·L-1;丁酰肼(B 9):0、2、4、6、8、10 mg·L-1)开展愈伤组织离体保存试验。在保存过程中,结合流式细胞术和SSR分子标记技术,检测不同保存代数下愈伤组织的细胞倍性和遗传稳定性,并对保存第15代的愈伤组织进行恢复生长试验。【结果】通过对6-BA+IBA浓度组合、渗透调节剂、生长抑制剂的筛选,改良MS+0.8 mg·L-16-BA+0.2 mg·L-1 IBA+45 g·L-1蔗糖+6.0 g·L-1琼脂粉(pH5.8)是适宜的青钱柳愈伤组织离体保存培养基,该培养基可有效地限制青钱柳愈伤组织生长;90天为适宜的继代保存时间,愈伤组织存活率为100%;愈伤组织呈黄绿色,颗粒状、颗粒小且疏松。与对照组(叶片)相比,不同保存代数下愈伤组织的细胞倍性没有发生改变,遗传稳定性好。保存的愈伤组织均能恢复生长,在形态、生活力和生长势方面表现好,并能够分化。【结论】适宜的青钱柳愈伤组织离体保存的培养基为改良MS基本培养基+0.8 mg·L-16-BA+0.2 mg·L-1 IBA+45 g·L-1蔗糖+6.0 g·L-1琼脂粉,pH5.8,在(20±1)℃下每隔90天转接1次,是青钱柳愈伤组织离体保存继代的最佳途径;保存的愈伤组织遗传稳定性好、未发生变异。甘露醇、肌醇、多效唑和丁酰肼等添加剂均不利于青钱柳愈伤组织的离体保存。

【Objective】This study was aimed to explore a method for in vitro conservation of callus by selecting osmotic regulators and growth inhibitor on the basis of the optimization of plant hormones in order to providing a theoretical foundation and technical support for in vitro conservation of germplasm resources in Cyclocarya paliurus.【Method】The callus of C.paliurus was used for in vitro conservation,the contents of 6-BA( 0.4,0.6,0.8,1.0 mg·L-1) +IBA( 0.2,0.4 mg·L-1) in the medium were optimized by using two-factor complete randomization.Sugar contents( 30,45,50,55,60 g·L-1) and number of days for conservation( 60,90,120,150,180 days),contents of osmoregulators( mannitol: 0,10,20,30,40,50 g·L-1;inositol: 0,10,20,30,40,50 g·L-1),contents of growth inhibitors( paclobutrazol( PP333) : 0,2,4,6,8,10 mg·L-1;daminozide( B9) : 0,2,4,6,8,10 mg·L-1) were tested in the in vitro conservation tests by using single-factor complete randomization.Cell ploidy and genetic stability of the callus at different conservation times were detected by flow cytometry and SSR markers,and the callus at the fifteenth generation( T15) was used for re-growth test.【Result】The results showed that the optimization of 6-BA + IBA content combination,and the selection of osmoregulator,growth inhibitor and number of conservation days,the improved MS medium with 0.8 mg·L-16-BA+0.2 mg·L-1 IBA+ 45 g·L-1 sugar + 6.0 g·L-1 agar( pH5.8) was suitable for in vitro conservation of the callus,effectively inhibiting the growth of callus.The suitable duration for callus subculture was 90 days,with a callus survival rate of 100%,and the callus was yellowish green in color,granular in shape,small and porous.Compared with the leaves used as control,no changes were found in cell ploidy and genetic stability in different generations of subculture.The calluses in vitro conservation were all able to regrow and differentiate in the medium,displaying good performance in morphology,viability and growth vigor. 【Conclusion】Our results demonstrated that the appropriate medium for callus in vitro conservation of C.paliurus was the improved MS medium with 0.8 mg·L-16-BA+0.2 mg·L-1 IBA +45 g·L-1 sugar+6.0 g·L-1 agar( pH5.8),and the optimal subculture is once every 90 days under( 20±1) ℃ . No variation was found in the callus in vitro conservation indicating good genetic stability.Use of additives of mannitol,inositol,PP333 and B9 was harmful to in vitro callus conservation of C.paliurus.